Agonists of Bitter Taste Receptors and Uses Thereof

ABSTRACT

The present invention relates to agonists of the human bitter-taste receptor hTAS2R7 and its role in bitter taste transduction. The invention also relates to assays for screening molecules that modulate, e.g. suppress or block hTAS2R7 bitter taste transduction or bitter taste response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/791,409, filed Mar.11, 2008, which is the U.S. national stage application of InternationalPatent Application No. PCT/EP2005/012385, filed Nov. 18, 2005, whichclaims the benefit of U.S. Provisional Patent Application No.60/629,035, filed Nov. 18, 2004, the disclosures of which are herebyincorporated by reference in their entireties, including all figures,tables and amino acid or nucleic acid sequences.

BACKGROUND

Investigators have recently turned their attention to understanding thebiological mechanisms of taste, and in particular bitter taste. For areview of the literature see, for example, Caicedo A. and Roper S. D.(2001) Science 291: 1557-1560; Dulac C. (2000) Cell 100: 607-610;Kinnamon S. C. (2000) Neuron 25: 507-510; Lindemann B. (2001) Nature413: 219-225; and Margolskee R F. (2001) J. Biol. Chem. 277: 1-4.

Bitter taste is aversive, and as such provides humans with a mechanismof protection against poisonous substances, which are generallybitter-tasting compounds. More subtly, bitter-tastants also affect thepalatability of food, beverages, thereby influencing human nutritionalhabits as is more fully discussed by Drewnowski in “The Science andComplexity of Bitter Taste”, (2001) Nutr. Rev. 59: 163-169. They alsoaffect the palatability of other ingestibles such as orally administeredpharmaceuticals and nutraceuticals. Understanding the mechanism ofbitter taste transduction has implications for the food andpharmaceutical industries. If the bitter taste transduction pathway canbe manipulated, it may be possible to suppress or eliminate bitter tasteto render foods more palatable and increase patient compliance with oralpharmaceutics.

Taste transduction involves the interaction of molecules, i.e. tastantswith taste receptor-expressing cells which reside in the taste budslocated in the papillae of the tongue. Taste buds relay information tothe brain on the nutrient content of food and the presence of poisons.Recent advances in biochemical and physiological studies have enabledresearchers to conclude that bitter taste transduction is mediated byso-called G-protein coupled receptors (GPCRs). GPCRs are 7 transmembranedomain cell surface proteins that amplify signals generated at a cellsurface when the receptor interacts with a ligand (a tastant) whereuponthey activate heterotrimeric G-proteins. The G-proteins are proteincomplexes that are composed of alpha and beta-gamma subunits. They areusually referred to by their alpha subunits and classified generallyinto 4 groups: G_(alpha s, i, q) and ₁₂. The G_(alpha q) type couplewith GPCRs to activate phospholipase C which leads to an increase incellular Ca²⁺. There are many G_(q)-type G-proteins that are promiscuousand can couple to GPCRs, including taste receptors, and these so-called“promiscuous” G-proteins are well known in the art. These G-proteinsdissociate into alpha and beta-gamma subunits upon activation, resultingin a complex cascade of cellular events that result in the cellproducing second messengers, such as calcium ions, that enable the cellsto send a signal to the brain indicating a bitter response.

There is also anatomical evidence that GPCRs mediate bitter tastetransduction: clusters of these receptors are found in mammalian tastecells containing gustducin. Gustducin is a G-protein subunit that isimplicated in the perception of bitter taste in mammals see, forexample, Chandrashekar, J. et al. (2000) Cell 100: 703-711; Matsunami H.et al. (2000) Nature 404: 601-604; or Adler E. et al. (2000) Cell 100:693-702. cDNAs encoding such GPCRs have been identified, isolated, andused as templates to compare with DNA libraries using insilicodata-mining techniques to identify other related receptors. In thismanner it has been possible to identify a family of related receptors,the so-called T2R or TAS2R family of receptors, that have beenputatively assigned as bitter receptors.

Humans are able to detect with a limited genetic repertoire of about 30receptor genes thousands of different bitter compounds. Since theirdiscovery in the year 2000 (Adler E. et al. (2000) supra; ChandrashekarJ. et al. (2000) supra; Matsunami H. et al (2000) supra) only fewmammalian TAS2Rs have been deorphanised, i.e. ligands, in particularagonists have been identified. The murine mTAS2R5 (Chandrashekar J. etal (2000) supra) and the rat rTAS2R9 (Bufe B. et al. (2002) NatureGenetics 32:397-401) respond to the toxic bitter substancecycloheximide, the mouse mTAS2R8 and the human hTAS2R4 respond to highdoses of denatonium and, to a lesser extend, to 6-n-propyl-2-thiouracil(Chandrashekar J. et al. (2000) supra), the human hTAS2R10 and hTAS2R16respond selectively to strychnine and bitter β-glucopyranosides,respectively (Bufe B. et al. (2002) supra). Although for some TAS2Rs alimited promiscuity (mTAS2R8, hTAS2R4) or specificity for a group ofchemically related compounds (hTAS2R16) was reported, the relativeselectivity of ligand recognition by the receptors published to datedoes, by far, not explain the enormous number of bitter tastantsrecognised by the mammalian gustatory system. There are several possiblemechanisms conceivable to increase the number of tastants recognised bya limited number of taste receptor genes, the simplest way would be tohave receptors which exhibit a broad tuning to a great number ofstructurally divergent ligands.

The present inventors now show that the human bitter receptors hTAS2R1and hTAS2R7 respond to two quite different bitter compounds and, thus,appear to be bitter taste receptors with such a broad tuning.Furthermore, the present inventors were able to show that hTAS2R3 andhTAS2R40 respond specifically to a particular bitter compound and, thus,might constitute examples for more specific bitter taste receptors. Thismakes the identification of antagonists for the hTAS2R1 and hTAS2R7receptors particularly attractive, since it can be envisioned that byblocking the hTAS2R1 and/or hTAS2R7 receptor the bitter perceptionelicited by a wide variety of different bitter tastants can be decreasedor blocked. In addition the deorphanization of hTAS2R3 and hTAS2R40 willallow to identify further agonists or antagonists for these particularbitter taste receptors.

DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

The present inventors have identified agonists for the hTAS2R1, hTAS2R3,hTAS2R7 and hTAS2R40 bitter taste receptors, and have found that theyrespond with specificity toward certain classes of bitter compounds thatare important in the food and pharmaceutical industries. The agonistsprovided by the present inventors enables the skilled person to designintelligent compound libraries to screen for antagonists to the bitterresponse of these receptors, which in turn enables the development ofcompounds and compositions to suppress or eliminate bitter tastingcomponents of foods, in particular animal foods, nutrients and dietarysupplements and pharmaceutical or homeopathic preparations containingsuch phyto-chemicals. Similarly, the invention also enables the skilledperson to screen for additional bitter ligands, or even to screen forcompounds that enhance a bitter response, such as might be useful in thefood industry.

Therefore, in one aspect the present invention provides a method forisolating an agonist or antagonist of hTAS2R1, hTAS2R3, hTAS2R7 andhTAS2R40 bitter taste receptor activity, respectively, wherein thehTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 bitter taste receptors areencoded by a polynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide encoding at least the mature form of the        polypeptide having the deduced amino acid sequence as shown in        SEQ ID NO: 2, 4, 6, or 8;    -   (b) a polynucleotide having the coding sequence, as shown in SEQ        ID NO: 1, 3, 5, or 7 encoding at least the mature form of the        hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 polypeptide,        respectively;    -   (c) polynucleotides encoding a fragment or derivative of a        polypeptide encoded by a polynucleotide of any one of (a) to        (b), wherein in said derivative one or more amino acid residues        are conservatively substituted compared to said polypeptide, and        said fragment or derivative has hTAS2R1, hTAS2R3, hTAS2R7 and        hTAS2R40, respectively, bitter taste receptor activity;    -   (d) polynucleotides which are at least 50% identical to a        polynucleotide as defined in any one of (a) to (c) and which        code for a polypeptide having hTAS2R1, hTAS2R3, hTAS2R7 and        hTAS2R40 bitter taste receptor activity, respectively; and    -   (e) polynucleotides the complementary strand of which        hybridizes, preferably under stringent conditions to a        polynucleotide as defined in any one of (a) to (d) and which        code for a polypeptide having hTAS2R1, hTAS2R3, hTAS2R7 and        hTAS2R40 bitter taste receptor activity, respectively;        comprising:    -   (1) contacting a polypeptide encoded by said polynucleotide, a        host cell genetically engineered with said polynucleotide or        with a vector containing said polynucleotide with a potential        antagonist;    -   (2) determining whether the potential antagonists antagonizes        the bitter taste receptor activity of said polypeptide        wherein prior, concomitantly and/or after step (1) said        polypeptide, said host cell or said vector is contacted with an        agonist selected from the group consisting of chloramphenicol,        humulone, and diphenylthiourea, picrotoxinin and yohimbine for        hTAS2R1, creatinine for hTAS2R3, creatinine and cromolyn for        hTAS2R7 and humulone for hTAS2R40 or agonistic derivatives of        the respective agonist thereof.

The polynucleotide employed in this method encodes a polypeptide thatstill exhibits essentially the same activity as the mature hTAS2R1,hTAS2R3, hTAS2R7 and hTAS2R40 bitter taste receptor, respectively, i.e.has “bitter taste receptor activity”. Preferably the polypeptide has atleast 20% (e.g., at least: 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%;98%; 99%; 99.5%; or 100% or even more) of the activity of thefull-length hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40, respectively. Onepreferred way of measuring hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40activity, respectively, is the ability to release intracellular calciumin a heterologous cell expression system like, for example,(HEK293T/G16gust44) cells that stably expresses a chimeric G-proteinconsisting of G_(α16) and 44 carboxylterminal amino acids ofα-gustducin, in response to bitter tastants, which is dependent on theexpression of polypeptides encoded by the polynucleotides of the presentinvention. The amount of intracellular calcium released can be monitoredby, for example, the in vitro FLIPR assay described herein but also bythe measurement of one of a variety of other parameters including, forexample, IP₃ or cAMP. Additional ways of measuring G-protein coupledreceptor activity are known in the art and comprise without limitationelectrophysiological methods, transcription assays, which measure, e.g.activation or repression of reporter genes which are coupled toregulatory sequences regulated via the respective G-protein coupledsignalling pathway, such reporter proteins comprise, e.g., CAT or LUC;assays measuring internalization of the receptor; or assays in frogmelanophore systems, in which pigment movement in melanophores is usedas a readout for the activity of adenylate cyclase or phospholipaseC(PLC), which in turn are coupled via G-proteins to exogenouslyexpressed receptors (see, for example, McClintock T. S. et al. (1993)Anal. Biochem. 209: 298-305; McClintock T. S. and Lerner M. R. (1997)Brain Res. Brain, Res. Protoc. 2: 59-68, Potenza M N (1992) Pigment CellRes. 5: 372-328, and Potenza M. N. (1992) Anal. Biochem. 206: 315-322)

The term “potential antagonist”, comprises any perceivable chemicalsubstance or combination thereof in a non-purified, partially purifiedor purified state, however, an antagonist of hTAS2R1, hTAS2R3, hTAS2R7and hTAS2R40 bitter taste receptor activity, respectively, is asubstance which lowers the hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 bittertaste receptor activity, respectively, determined in the presence of therespective agonist by at least 10% (e.g., at least: 10%, 15%; 20%; 30%;40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100%) oncecontacted with the bitter taste receptor. Preferably the antagonistexerts this action when it is contacted prior, concomitantly or after,preferably concomitantly, the contacting the hTAS2R1, hTAS2R3, hTAS2R7and hTAS2R40 polypeptide, respectively, the host cell expressing thehTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 polypeptide, respectively, or thevector comprising the hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40polypeptide, respectively, with one of the identified hTAS2R1, hTASR3,hTASR7 and hTASR40 agonists, respectively.

The hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 polynucleotide molecules,respectively, usable in the method of the present invention can be DNA,cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded, the sense and/or an antisense strand. Segments of thesemolecules are also considered within the scope of the invention, and canbe produced by, for example, the polymerase chain reaction (PCR) orgenerated by treatment with one or more restriction endonucleases. Aribonucleic acid (RNA) molecule can be produced by in vitrotranscription.

The polynucleotide molecules useable in the method of the presentinvention can contain naturally occurring sequences, or sequences thatdiffer from those that occur naturally, but, due to the degeneracy ofthe genetic code, encode the same polypeptide (for example, thepolypeptide with SEQ ID NO: 2, 4, 6, or 8). In addition, these nucleicacid molecules are not limited to coding sequences, e.g., they caninclude some or all of the non-coding sequences that lie upstream ordownstream from a coding sequence.

The polynucleotide molecules of the invention can be synthesized invitro (for example, by phosphoramidite-based synthesis) or obtained froma cell, such as the cell of a bacteria or a mammal. The nucleic acidscan be those of a human but also derived from a non-human primate,mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat aslong as they fulfill the criteria set out above. Combinations ormodifications of the nucleotides within these types of nucleic acids arealso encompassed.

In addition, the polynucleotides useable in the method of the presentinvention can encompass segments that are not found as such in thenatural state. Thus, the invention encompasses recombinant nucleic acidmolecules incorporated into a vector (for example, a plasmid or viralvector) or into the genome of a heterologous cell (or the genome of ahomologous cell, at a position other than the natural chromosomallocation). Recombinant nucleic acid molecules and uses therefore arediscussed further below.

In certain preferred embodiments the method of the present inventionuses isolated nucleic acid molecules which are at least 50% (or 55%,65%, 75%, 85°/a, 95%, or 98%) identical to: (a) a nucleic acid moleculethat encodes the polypeptide of SEQ ID NO: 2, 4, 6 or 8; (b) thenucleotide sequence of SEQ ID NO: 1, 3, 5, or 7 and (c) a nucleic acidmolecule which includes a segment of at least 30 (e.g., at least 30, 40,50, 60, 80, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800,850, and 900) nucleotides of SEQ ID NO: 1, 3, 5 and 7, respectively.

The determination of percent identity between two sequences isaccomplished using the mathematical algorithm of Karlin and Altschul(1993) Proc. Natl. Acad. Set. USA 90: 5873-5877. Such an algorithm isincorporated into the BLASTN and BLASTP programs of Altschul et al.(1990) J. Mol. Biol. 215: 403-410. BLAST nucleotide searches areperformed with the BLASTN program, score=100, word length=12, to obtainnucleotide sequences homologous to hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40encoding nucleic acids. BLAST protein searches are performed with theBLASTP program, score=50, wordlength=3, to obtain amino acid sequenceshomologous to the hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 polypeptide,respectively. To obtain gapped alignments for comparative purposes,Gapped BLAST is utilized as described in Altschul et al. (1997) NucleicAcids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs are used.

Hybridization can also be used as a measure of homology between twonucleic acid sequences. A nucleic acid sequence encoding hTAS2R1,hTAS2R3, hTAS2R7 or hTAS2R40, or a portion thereof, can be used as ahybridization probe according to standard hybridization techniques. Thehybridization of a hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40 probe to DNA orRNA from a test source (e.g., a mammalian cell) is an indication of thepresence of the hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40 DNA or RNA in thetest source. Hybridization conditions are known to those skilled in theart and can be found, for example, in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Moderatehybridization conditions are defined as equivalent to hybridization in2× sodium chloride/sodium citrate (SSC) at 30° C., followed by a wash in1×SSC, 0.1% SDS at 50° C. Highly stringent conditions are defined asequivalent to hybridization in 6× sodium chloride/sodium citrate (SSC)at 45° C., followed by a wash in 0.2×SSC, 0.1% SDS at 65° C.

The polynucleotides or proteins useable in the method of the presentinvention can be comprised in a vector containing the polynucleotide(s)or a protein encoded by above-mentioned polynucleotide. The term“vector” refers to a protein or a polynucleotide or a mixture thereofwhich is capable of being introduced or of introducing the proteinsand/or nucleic acid comprised therein into a cell. It is preferred thatthe proteins encoded by the introduced polynucleotide are expressedwithin the cell upon introduction of the vector.

In a preferred embodiment a vector useable in the method of the presentinvention comprises plasmids, phagemids, phages, cosmids, artificialmammalian chromosomes, knock-out or knock-in constructs, viruses, inparticular adenoviruses, vaccinia viruses, attenuated vaccinia viruses,canary pox viruses, lentivirus (Chang, L. J. and Gay, E. E. (2001) Curr.Gene Therap. 1: 237-251), herpes viruses, in particular Herpes simplexvirus (HSV-1, Carlezon, W. A. et al. (2000) Crit. Rev. Neurobiol. 14:47-67), baculovirus, retrovirus, adenoassociated-virus (AAV, Carter, P.J. and Samulski, R. J. (2000) J. Mol. Med. 6:17-27), rhinovirus, humanimmune deficiency virus (HIV), filovirus and engineered versions thereof(see, for example, Cobinger G. P. et al. (2001) Nat. Biotechnol.19:225-30), virosomes, “naked” DNA liposomes, and nucleic acid coatedparticles, in particular gold spheres. Particularly preferred are viralvectors like adenoviral vectors or retroviral vectors (Lindemann et al.(1997) Mol. Med. 3: 466-76 and Springer et al. (1998) Mol. Cell. 2:549-58). Liposomes are usually small unilamellar or multilamellarvesicles made of cationic, neutral and/or anionic lipids, for example,by ultrasound treatment of liposomal suspensions. The DNA can, forexample, be ionically bound to the surface of the liposomes orinternally enclosed in the liposome. Suitable lipid mixtures are knownin the art and comprise, for example, DOTMA (1, 2-Dioleyloxpropyl-3-trimethylammoniumbromid) and DPOE (Dioleoylphosphatidyl-ethanolamin) whichboth have been used on a variety of cell lines.

Nucleic acid coated particles are another means for the introduction ofnucleic acids into cells using so called “gene guns”, which allow themechanical introduction of particles into cells. Preferably theparticles itself are inert, and therefore, are in a preferred embodimentmade out of gold spheres.

In a further aspect polynucleotides useable in the method of the presentinvention are operatively linked to expression control sequencesallowing expression in prokaryotic and/or eukaryotic host cells. Thetranscriptional/translational regulatory elements referred to aboveinclude but are not limited to inducible and non-inducible,constitutive, cell cycle regulated, metabolically regulated promoters,enhancers, operators, silencers, repressors and other elements that areknown to those skilled in the art and that drive or otherwise regulategene expression. Such regulatory elements include but are not limited toregulatory elements directing constitutive expression like, for example,promoters transcribed by RNA polymerase III like, e.g. promoters for thesnRNA U6 or scRNA 7SK gene, the cytomegalovirus hCMV immediate earlygene, the early or late promoters of SV40 adenovirus, viral promoter andactivator sequences derived from, e.g. NBV, HCV, HSV, HPV, EBV, HTLV,MMTV or HIV; which allow inducible expression like, for example, CUP-1promoter, the tet-repressor as employed, for example, in the tet-on ortet-off systems, the lac system, the trp system; regulatory elementsdirecting tissue specific expression, preferably taste bud specificexpression, e.g. PLCβ2 promoter or gustducin promoter, regulatoryelements directing cell cycle specific expression like, for example,cdc2, cdc25C or cyclin A; or the TAC system, the TRC system, the majoroperator and promoter regions of phage A, the control regions of fd coatprotein, the promoter for 3-phosphoglycerate kinase, the promoters ofacid phosphatase, and the promoters of the yeast α- or α-mating factors.

As used herein, “operatively linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

Similarly, the polynucleotides useable in the method of the presentinvention can form part of a hybrid gene encoding additional polypeptidesequences, for example, a sequence that functions as a marker orreporter. Examples of marker and reporter genes include β-lactamase,chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo^(r), G418^(r)), dihydrofolatereductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidinekinase (TK), lacZ (encoding β-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the method of the invention,skilled artisans will be aware of additional useful reagents, forexample, additional sequences that can serve the function of a marker orreporter.

The method of the present invention may also use hybrid polypeptides orpolynucleotides encoding them. In general a hybrid polypeptide willinclude a first portion and a second portion; the first portion beingone or more hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40 polypeptide and thesecond portion being, for example, the reporter(s) described above or anIg constant region or part of an Ig constant region, e.g., the CH2 andCH3 domains of IgG2a heavy chain. Other hybrids could include anantigenic tag or His tag to facilitate purification and/or detection.Recombinant nucleic acid molecules can also contain a polynucleotidesequence encoding the hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40 polypeptideoperatively linked to a heterologous signal sequence. Such signalsequences can direct the protein to different compartments within thecell and are well known to someone of skill in the art. A preferredsignal sequence is a sequence that facilitates secretion of theresulting protein.

Another aspect of the present invention is the use of a host cellgenetically engineered with a polynucleotide or a vector as outlinedabove. The host cells that may be used in the method of the presentinvention include but are not limited to prokaryotic cells such asbacteria (for example, E. coli and B. subtilis), which can betransformed with, for example, recombinant bacteriophage DNA, plasmidDNA, or cosmid DNA expression vectors containing the polynucleotidemolecules of the invention; simple eukaryotic cells like yeast (forexample, Saccharomyces and Pichia), which can be transformed with, forexample, recombinant yeast expression vectors containing thepolynucleotide molecule of the invention; insect cell systems like, forexample, Sf9 or Hi5 cells, which can be infected with, for example,recombinant virus expression vectors (for example, baculovirus)containing the polynucleotide molecules; Xenopus oocytes, which can beinjected with, for example, plasmids; plant cell systems, which can beinfected with, for example, recombinant virus expression vectors (forexample, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV))or transformed with recombinant plasmid expression vectors (for example,Ti plasmid) containing a hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40nucleotide sequence; or mammalian cell systems (for example, COS, CHO,BHK, HEK293, VERO, HeLa, MDCK, Wi38, and NIH 3T3 cells), which can betransformed with recombinant expression constructs containing, forexample, promoters derived, for example, from the genome of mammaliancells (for example, the metallothionein promoter) from mammalian viruses(for example, the adenovirus late promoter and the vaccinia virus 7.5Kpromoter) or from bacterial cells (for example, the tet-repressorbinding is employed in the tet-on and tet-off systems). Also useful ashost cells are primary or secondary cells obtained directly from amammal and transfected with a plasmid vector or infected with a viralvector. Depending on the host cell and the respective vector used tointroduce the polynucleotide of the invention the polynucleotide canintegrate, for example, into the chromosome or the mitochondrial DNA orcan be maintained extrachromosomally like, for example, episomally orcan be only transiently comprised in the cells.

In a preferred embodiment, the hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40expressed by such cells are functional and have bitter taste receptoractivity, i.e., upon binding to one or more bitter molecules theytrigger an activation pathway in the cell. The cells are preferablymammalian (e.g., human, non-human primate, horse, bovine, sheep, pig,dog, cat, goat, rabbit, mouse, rat, guinea pig, hamster, or gerbil)cells, insect cells, bacterial cells, or fungal (including yeast) cells.

The polypeptides useable in the method of the invention include allthose disclosed herein and functional fragments of these polypeptides.The terms “polypeptide” and “protein” are used interchangeably and meanany peptide-linked chain of amino acids, regardless of length orposttranslational modification. As used herein, a functional fragment ofthe hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40 is a fragment of the hTAS2R1,hTAS2R3, hTAS2R7 or hTAS2R40 that is shorter than the full-lengthhTAS2R1, hTASR3, hTASR7 or hTASR40 but that has at least 20% (e.g., atleast: 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or100% or even more) of the ability of the full-length hTAS2R1, hTAS2R3,hTAS2R7 or hTAS2R40 to be stimulated by one of the bitter substancesidentified herein. Binding assays and bitter substances are described inmore detail herein below. The polypeptides can also include fusionproteins that contain either a full-length hTAS2R1, hTAS2R3, hTAS2R7 orhTAS2R40 polypeptide or a functional fragment of it fused to anunrelated amino acid sequence. The unrelated sequences can add furtherfunctional domains or signal peptides.

The polypeptides can be any of those described above but with not morethan 50 (e.g., not more than: 50, 45, 40, 35, 30, 25, 20, 15, 14, 13,12, 11, 10, nine, eight, seven, six, five, four, three, two, or one)conservative substitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine. All that is required of a polypeptide havingone or more conservative substitutions is that it has at least 20%(e.g., at least: 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%;99.5%; or 100% or even more) of the ability of the full-length hTAS2R1,hTAS2R3, hTAS2R7 or hTAS2R40 to be stimulated by the respective bittersubstance.

Polypeptides and fragments of the polypeptides useable in the method ofthe present invention can be modified, for example, for in vivo use bythe addition of blocking agents, at the amino- and/or carboxyl-terminalends, to facilitate survival of the relevant polypeptide in vivo. Thiscan be useful in those situations in which the peptide termini tend tobe degraded by proteases prior to cellular uptake. Such blocking agentscan include, without limitation, additional related or unrelated peptidesequences that can be attached to the amino and/or carboxyl terminalresidues of the peptide to be administered. This can be done eitherchemically during the synthesis of the peptide or by recombinant DNAtechnology by methods familiar to artisans of average skill.

The antagonists or agonists of the bitter taste receptors identifiedherein are of great importance for specific stimulation of a givenbitter taste receptor and identification of substances that antagonizeit, respectively.

The term “contacting” in the context of the present invention means anyinteraction between the antagonist and/or agonist with the polypeptideor the host cell, whereby any of the at least two components can beindependently of each other in a liquid phase, for example in solution,or in suspension or can be bound to a solid phase, for example, in theform of an essentially planar surface or in the form of particles,pearls or the like. In a preferred embodiment a multitude of differentcompounds are immobilized on a solid surface like, for example, on acompound library chip and the protein of the present invention issubsequently contacted with such a chip. In another preferred embodimentthe host cells are genetically engineered with a polynucleotide encodinghTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40 or with a vector containing such apolynucleotide, express the hTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40 bittertaste receptor at the cell surface and are contacted separately in smallcontainers, e.g., microtitre plates, with various compounds.

The term “agonistic derivatives thereof” refers to agonists, which arederived from the respectively indicated agonist, i.e. bitter substance,by chemical modification and which elicit at least 20% (e.g., at least:20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100% oreven more) of the bitter taste receptor activity, if compared to therespective unmodified bitter substance. Chemical modification includeswithout limitation the introduction of one or more, preferably two,three or four novel side chains or residues or the exchange of one ormore functional groups like, for example, introduction or exchange of H;linear or branched alkyl, in particular lower alkyl (C₁, C₂, C₃, C₄, andC₅, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-pentyl or iso-pentyl); substituted linear or branchedalkyl, in particular lower substituted alkyl; linear or branchedalkenyl, in particular lower alkenyl (C₂, C₃, C₄ and C₅, e.g. ethenyl,1-propenyl, 2-propenyl, iso-propenyl, 1-butenyl, 2-butenyl, 3-butenyl;substituted linear or branched alkenyl, in particular lower substitutedalkenyl; linear or branched alkinyl, in particular lower alkinyl (C₂,C₃, C₄ and C₅); substituted linear or branched alkinyl, in particularlower substituted alkinyl; linear or branched alkanol, in particularlower alkanol (C₂, C₃, C₄, and C₅); linear or branched alkanal, inparticular lower alkanal (C₁, C₂, C₃, C₄, and C₅, e.g. COH, CH₂COH,CH₂CH₂COH; linear or branched alkyl carboxyl group, in particular loweralkyl carboxyl group (C₁, C₂, C₃, C₄ and C₅, e.g. formic, acetic,propionic, butyric, pentanoic acid group); substituted linear orbranched alkyl carboxyl groups, in particular lower alkyl carboxyl group(C₂, C₃, C₄ and C_(s), e.g. 2-methyl acetic acid, acetic acid, propionicacid, butyric acid, pentanoic acid group); aryl, in particular phenyl;substituted aryl, in particular substituted aryl; heteroaryl;substituted heteroaryl; alkylaryl, in particular benzyl; substitutedalkylaryl; in particular substituted benzyl; alkylheteroaryl;substituted alkylheteroaryl; aminoalkyl, C₁, C₂, C₃, C₄ and C₅, e.g.—NHCH₃, —NHCH₂CH₃, —N(CH₃)₂; substituted aminoalkyl; aminoketone, inparticular —NHCOCH₃; substituted aminoketone; aminoaryl, in particular—NH-Ph; substituted aminoaryl, in particular substituted —NH-Ph; CN;NH₂; Halogen, in particular F, Cl, and Br; NO₂; OH; SH; NH; CN; or COOHgroup. If the residues mentioned above are substituted they arepreferably mono, di, or tri substituted with a substituent selected fromthe group of halogen, in particular F, Cl, and Br, NH₂, NO₂, OH, SH, NH,CN, aryl, alkylaryl, heteroaryl, alkylheteroaryl, COH or COOH. Thevarious chemically modified agonists can be assessed for their activityin any of the assay systems described herein.

As a further step after measuring the antagonizing effect of a potentialantagonist and after having measured the decrease of bitter taste for atleast two different potential antagonists at least one potentialantagonist can be selected, for example, on grounds of the detecteddecrease of intracellular release of calcium, if compared to contactingwith the known agonist alone.

The thus selected (potential) antagonist is than in a preferredembodiment modified in a further step. Modification can be effected by avariety of methods known in the art, which include without limitationthe introduction of one or more, preferably two, three or four novelside chains or residues or the exchange of one or more functional groupslike, for example, introduction or exchange of halogens, in particularF, Cl or Br; the introduction or exchange of lower alkyl residues,preferably having one to five carbon atoms like, for example, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl oriso-pentyl residues; lower alkenyl residues, preferably having two,three, four or five carbon atoms; lower alkinyl residues, preferablyhaving two, three, four or five carbon atoms, which can in a preferredembodiment be further substituted with F, Cl, Br, NH₂, NO₂, OH, SH, NH,CN, aryl, heteroaryl. COH or COOH group; or the introduction of, forexample, one or more residue(s) selected from the group consisting ofNH₂, NO₂, OH, SH, NH, CN, aryl, alkylaryl, heteroaryl, alkylheteroaryl,COH or COOH group.

The thus modified (potential) antagonists are than individually testedwith the method of the present invention, i.e. they are contacted withthe polypeptide as such or with the polypeptide expressed in a hostcell, which has been contacted prior, concomitantly or after step (1)with one of the indentified agonists or derivatives thereof andsubsequently activation of the bitter taste receptor activity by themodified antagonists is measured. The activation of the hTAS2R1,hTAS2R3, hTAS2R7 or hTAS2R40 protein can be measured, e.g. by theintracellular calcium release mediated. If needed the steps of selectingthe antagonist, modifying the compound, contacting the antagonist with apolypeptide or a host cell and measuring of the activation of the bittertaste receptor activity can be repeated a third or any given number oftimes as required. The above described method is also termed “directedevolution” of an antagonist since it involves a multitude of stepsincluding modification and selection, whereby antagonizing or agonizingcompounds are selected in an “evolutionary” process optimizing theircapabilities with respect to a particular property, e.g. their abilityto inhibit, activate or modulate the activity of hTAS2R1, hTAS2R3,hTAS2R7 or hTAS2R40, in particular inhibit or stimulate theintracellular release of calcium.

In order to express cDNAs encoding the receptors, one typicallysubclones receptor cDNA into an expression vector that contains a strongpromoter to direct transcription, a transcription/translationterminator, and a ribosome-binding site for translational initiation.Suitable bacterial promoters are well known in the art, e.g., E. coli,Bacillus sp., and Salmonella, and kits for such expression systems arecommercially available. Similarly eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available. The eukaryotic expression vector maybe, for example an adenoviral vector, an adeno-associated vector, or aretroviral vector.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the receptor-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operatively linked to the nucleic acid sequence encoding thereceptor and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding the receptor may typically be linked to amembrane-targeting signal such as the N-terminal 45 amino acids of therat somatostatin receptor 3 sequence to promote efficient cell-surfaceexpression of the recombinant receptor. Additional elements of thecassette may include, for example enhancers.

An expression cassette should also contain a transcription terminationregion down-stream of the structural gene to provide for efficienttermination. The termination region may be obtained from the same geneas the promoter sequence or may be obtained from different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ, but there are many more knownin the art to the skilled person that can be usefully employed.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g. SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMT010/A′, pMAMneo-5, baculovirus pDSVE, pcDNA3.1, pIRES and any othervector allowing expression of proteins under the direction of the SV40early promoter, SV40 late promoter, metallothionein promoter, murinemammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrinpromoter, or other promoters shown effective for expression ineukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding drugresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particular drugresistance gene chosen is not critical, any of the many drug resistancegenes known in the art are suitable. The prokaryotic sequences areoptionally chosen such that they do not interfere with the replicationof the DNA in eukaryotic cells, if necessary.

Standard transfection methods can be used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofthe receptor, which are then purified using standard techniques.

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell. It isonly necessary that the particular genetic engineering procedure used becapable of successfully introducing at least one gene into the host cellcapable of expressing the receptor.

After the expression vector is introduced into the cells, thetransfected cells may be cultured under conditions favouring expressionof the receptor, which is recovered from the culture using standardtechniques. For example the cells may be burst open either mechanicallyor by osmotic shock before being subject to precipitation andchromatography steps, the nature and sequence of which will depend onthe particular recombinant material to be recovered. Alternatively, therecombinant protein may be recovered from the culture medium in whichthe recombinant cells had been cultured.

The activity of the receptor described herein can be assessed using avariety of in vitro and in vivo assays to determine functional,chemical, and physical effects, e.g., measuring ligand binding,secondary messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺) ion flux,phosphorylation levels, transcription levels, of reporter constructsneurotransmitter levels, and the like. Such assays are used in themethod of the present invention to test for the activity of thereceptors.

Samples or assays that are treated with a potential receptor agonist maybe compared to control samples without the test compound (agonist orantagonist), to examine the extent of modulation. Control samples(treated with agonists only) are assigned a relative receptor activityvalue of 100. Inhibition of receptor activity is achieved when thereceptor activity value relative to the control is lower, and converselyreceptor activity is enhanced when activity relative to the control ishigher in the presence of identical amounts of the respective agonist.

The effects of the test compounds upon the function of the receptors canbe measured by examining any of the parameters described above. Anysuitable physiological change that affects receptor activity can be usedto assess the influence of a test compound on the receptors of thisinvention. When the functional consequences are determined using intactcells or animals, one can measure a variety of effects such as changesin intracellular secondary messengers such as Ca²⁺, IP₃ or cAMP.

Preferred assays for G-protein coupled receptors include cells that areloaded with ion sensitive dyes to report receptor activity. In assaysfor identifying modulatory compounds, changes in the level of ions inthe cytoplasm or membrane voltage will be monitored using an ionsensitive or membrane voltage fluorescent indicator, respectively. ForG-protein coupled receptors, promiscuous G-proteins such as G_(α15) andG_(α16) and chimeric G-proteins can be used in the assay of choice (see,for example, Wilkie et al. (1991) Proc. Nat. Acad. Sci. USA 88:10049-10053). Such promiscuous G-proteins allow coupling of a wide rangeof receptors to G-protein dependent signal pathways.

Receptor activation typically initiates subsequent intracellular events,e.g. increases in second messengers such as IP₃, which releasesintracellular stores of calcium ions. Activation of some G-proteincoupled receptors stimulates the formation of inositol trisphosphatethrough phospholipase C-mediated hydrolysis of phosphatidylinositolbisphosphate (Berridge & Irvine (1984) Nature 312: 315-21). IP₃ in turnstimulates the release of intracellular calcium ion stores. Thus, achange in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP₃ can be used to assess G-protein coupledreceptor function. Cells expressing such G-protein coupled receptors mayexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it may be desirable, although not necessary, to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

In a preferred embodiment, receptor activity is measured by expressingthe hTAS2R1, hTAS2R3, hTAS2R7, or hTAS2R40 receptors in a heterologouscell with a promiscuous G-protein, such as G_(α15), G_(α16), or achimeric G-protein that links the receptor to a phospholipase C signaltransduction pathway. A preferred cell line is HEK-293, although othermammalian cell lines are also preferred such as CHO and COS cells.Modulation of taste transduction is assayed by measuring changes inintracellular Ca²⁺ levels, which change in response to modulation of thereceptor signal transduction pathway via administration of a moleculethat associates with the receptor. Changes in Ca²⁺ levels are optionallymeasured using fluorescent Ca²⁺ indicator dyes and fluorometric imaging.

The activity of the signalling molecule and the increase or decrease ofthat activity in response to the potential agonist or antagonist can bedetermined as outlined above with respect to the identification ofbitter receptor taste activity. The respectively indicated percentincreases or decreases of the activity, which are required to qualify asantagonist or agonist do apply mutatis mutandis. Additionally the term“contacting” has the meaning as outlined above. Preferably thesignalling molecule and/or the promiscuous G-protein has been introducedinto the cell. The types of cell lines, which are preferred are thoseindicated above.

In yet another embodiment, the ligand-binding domains of the receptorscan be employed in vitro in soluble or solid-state reactions to assayfor ligand binding. Ligand binding to a receptor, or a domain of areceptor, can be tested in solution, in a bilayer membrane attached to asolid phase, in a lipid monolayer or vesicles. Thereby, the binding of amodulator to the receptor, or domain, can be observed using changes inspectroscopic characteristics, e.g. fluorescence, absorbance orrefractive index; or hydrodynamic (e.g. shape), chromatographic, orsolubility properties, as is generally known in the art.

The compounds tested as modulators of the receptors can be any smallchemical compound, or a biological entity, such as a protein, sugar,nucleic acid or lipid. Typically, test compounds will be small chemicalmolecules. Essentially any chemical compound can be used as a potentialmodulator or ligand in the assays of the invention, although knowledgeof the ligand specificity of an individual receptor would enable theskilled person to make an intelligent selection of interestingcompounds. The assays may be designed to screen large chemical librariesby automating the assay steps and providing compounds from anyconvenient source to assays, which are typically run in parallel (e.g.,in microtiter formats on microtiter plates in robotic assays). Theskilled person will understand that there are many suppliers oflibraries of chemical compounds.

Assays may be run in high throughput screening methods that involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic, or tastant compounds (that arepotential ligand compounds). Such libraries are then screened in one ormore assays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve as leadcompounds to further develop modulators for final products, or canthemselves be used as actual modulators.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art and no more needs to be stated here.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100 to about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 different compoundsis possible using the integrated systems of the invention.

Antagonists found by assay technology herein above described, ordevelopment compounds formed from such antagonists can be administereddirectly to a human subject to modulate bitter taste. Alternatively,such compounds can be formulated with other ingredients of preparationsto be taken orally, for example, foods, including animal food, andbeverages, pharmaceutical or nutraceutical or homeopathic preparations.

Therefore, another aspect of the invention is a process for theproduction of foodstuffs or any precursor material or additive employedin the production of foodstuffs comprising the steps of the abovedescribed processes for the identification of an antagonist of hTAS2R1,hTAS2R3, hTAS2R7 or hTAS2R40 and the subsequent step of admixing theidentified antagonist with foodstuffs or any precursor material oradditive employed in the production of foodstuffs.

Bitter taste is a particular problem when orally administeringpharmaceuticals, which often have an unpleasant bitter taste. Inparticular in elderly persons, children and chronically ill patientsthis taste can lead to a lack of compliance with a treatment regimen. Inaddition in veterinary applications the oral administration of bittertasting pharmaceuticals can be problematic. Therefore, a further aspectof the invention is a process for the production of a nutraceutical orpharmaceutical composition comprising the step of the process toidentify an antagonist or an agonist of hTAS2R1, hTAS2R3, hTAS2R7 andhTAS2R40 and the subsequent step of formulating the antagonist with anactive agent in a pharmaceutically acceptable form.

Consequently, a further aspect of the invention is a foodstuff, inparticular animal food, or any precursor material or additive employedin the production of foodstuffs produced according to the method of theinvention.

Also comprised is a nutraceutical or pharmaceutical composition producedaccording to the method of the invention and at least one active agentand optionally a pharmaceutically acceptable carrier and/or adjuvants.

The amount of compound to be taken orally must be sufficient to affect abeneficial response in the human subject, and will be determined by theefficacy of the particular taste modulators and the existence, nature,and extent of any adverse side-effects that accompany the administrationof a particular compound.

A further aspect of the present invention is the use of an agonist ofhTAS2R1, hTAS2R3, hTAS2R7 or hTAS2R40 activity selected from the groupconsisting of chloramphenicol, humulone, diphenylthiourea, picrotoxininand yohimbine for hTAS2R1, creatinine for hTAS2R3, creatinine andcromolyn for hTAS2R7 and humulone for hTAS2R40 and agonistic derivativesthereof to enhance bitter taste.

A further aspect of the present invention is the use of an antagonist ofhTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 activity selected from the groupof antagonistic derivatives of chloramphenicol, humulone,diphenylthiourea, picrotoxinin and yohimbine for hTAS2R1, creatinine forhTAS2R3, creatinine and cromolyn for hTAS2R7 and humulone for hTAS2R40to suppress bitter taste.

The term “antagonistic derivatives” of the respectively bitter tastantis a substance, which is derived from the respectively indicatedagonist, i.e. bitter substance, by chemical modification and whichlowers the hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 bitter taste receptoractivity, respectively, determined in the presence of the respectiveagonist, by at least 10% (e.g. at least 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or 100%) once contacted with abitter taste receptor. Preferably the antagonistic derivative exertsthis action, when it is contacted prior, concomitantly or after,preferably concomitantly, to the contacting of the hTAS2R1, hTAS2R3,hTAS2R7 and hTAS2R40 polypeptide, respectively, the host cell expressingthe hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40 polypeptide, respectively, orthe vector comprising the hTAS2R1, hTAS2R3, hTAS2R7 and hTAS2R40polypeptide, respectively with the agonist.

Chemical modification includes without limitation the introduction ofone or more, preferably two, three or four novel side chains or residuesor the exchange of one or more functional groups like, for example,introduction or exchange of H; linear or branched alkyl, in particularlower alkyl (C₁, C₂, C₃, C₄, and C₅, e.g. methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl or iso-pentyl);substituted linear or branched alkyl, in particular lower substitutedalkyl; linear or branched alkenyl, in particular lower alkenyl (C₂, C₃,C₄ and C₅, e.g. ethenyl, 1-propenyl, 2-propenyl, iso-propenyl,1-butenyl, 2-butenyl, 3-butenyl; substituted linear or branched alkenyl,in particular lower substituted alkenyl; linear or branched alkynyl, inparticular lower alkynyl (C₂, C₃, C₄ and C₅); substituted linear orbranched alkynyl, in particular lower substituted alkinyl; linear orbranched alkanol, in particular lower alkanol (C₂, C₃, C₄, and C₅);linear or branched alkanal, in particular lower alkanal (C_(I), C₂, C₃,C₄, and C₅, e.g. COH, CH₂COH, CH₂CH₂COH; linear or branched alkylcarboxyl group, in particular lower alkyl carboxyl group (C₁, C₂, C₃, C₄and C₅, e.g. formic, acetic, propionic, butyric, pentanoic acid group);substituted linear or branched alkyl carboxyl groups, in particularlower alkyl carboxyl group (C₂, C₃, C₄ and C₅, e.g. 2-methyl aceticacid, acetic acid, propionic acid, butyric acid, pentanoic acid group);aryl, in particular phenyl; substituted aryl, in particular substitutedaryl; heteroaryl; substituted heteroaryl; alkylaryl, in particularbenzyl; substituted alkylaryl; in particular substituted benzyl;alkylheteroaryl; substituted alkylheteroaryl; aminoalkyl, C₁, C₂, C₃, C₄and C₅, e.g.—NHCH₃, —NHCH₂CH₃, —N(CH₃)₂; substituted aminoalkyl;aminoketone, in particular —NHCOCH₃; substituted aminoketone; aminoaryl,in particular —NH-Ph; substituted aminoaryl, in particular substituted—NH-Ph; CN; NH₂; Halogen, in particular F, Cl, and Br; NO₂; OH; SH; NIT;CN; or COOH group.

The following figures and examples are merely illustrative of thepresent invention and should not be construed to limit the scope of theinvention as indicated by the appended claims in any way.

BRIEF DESCRIPTION OF THE TABLES AND FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

Table 1: Response of hTAS2R1 to chloramphenicol, humulone,diphenylthiourea, picrotoxinin and yohimbine.

Table 2 Response of hTAS2R3 to creatinine.

Table 3 Response of hTAS2R7 to creatinine and cromolyn.

Table 4 Response of hTAS2R40 to humulone.

FIGS. 1A-1D Expression and cell surface localisation of hTAS2R1 (FIG.1A), hTASR3 (FIG. 1B), hTASR7 (FIG. 1C) and hTASR40 (FIG. 1D) in HEK 293cells. Indirect immunocytochemistry of the TAS2R receptors (shown inlight grey) using monoclonal anti-HSV antibody (Novagen) and secondaryanti-mouse IgG antibody coupled to Alexa488 (Molecular Probes). Cellsmembrane (shown in dark grey) was stained with Avidin-D-Texas-Red.Yellow colour shows the colocalisation of the receptor with the cellsurface.

FIGS. 2A-2B Dose-response curves of hTAS2R1 for humulone andchloramphenicol. Different concentrations of humulone (FIG. 2A) andchloramphenicol (FIG. 2B) were applied onto hTAS2R1 transfected cells.

FIG. 3 Dose-response curves of hTAS2R7 for cromolyn. Differentconcentrations of cromolyn were applied onto hTAS2R7 transfected cells.

EXAMPLES Conditional Expression of hTAS2R1, hTASR3, hTASR 7 and hTASR40

Ligand screening using an automated fluorometric imaging plate reader(Molecular Devices) was essentially done as described (Bufe B. et al.(2002) Nature Genetics 32: 397-401). Briefly: The cells were seeded at adensity of 70,000±10,000 cells per well in 96-well microtiter plates.The cDNA of hTAS2R1, hTASR3, hTASR7 and hTASR40 supplemented with anamino terminal export tag corresponding to amino acids 1-45 of ratsomatostatin receptor 3 and a carboxy terminal HSV-tag was transientlytransfected into HEK-293T cells stably expressing the chimeric G-proteinsubunit G_(α16gust44) (Ueda T. et al. (2003) J. Neurosci. 23: 7376-7380)using Lipofectamine2000 (Invitrogen). Expression rates were determinedto be 20% for hTAS2R1; 35% for hTAS2R3, 14% for hTAS2R7 and 29% forhTAS2R40 by indirect immunocyclochemistry using monoclonal anti-HSVantibodies (Novagen) and secondary anti-mouse IgG antibodies coupled toAlexa 488 (Molecular probes) see (FIG. 1). 24-32 hours aftertransfection, the cells were then loaded for 1 hour with the calciumsensitive dye Fluo4-AM (2 μg/ml in DMEM, Molecular Probes). Cells werewashed 3× in solution C1 (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mMCaCl₂, and 10 mM Glucose, pH 7.4). Calcium mobilization was monitored byan automated fluorometric imaging plate reader (Molecular Devices).Ligands (Sigma-Aldrich) were dissolved in C1 solution. All data werecollected from experiments carried out in triplicate. For dose-responsecurves, the obtained calcium signals were corrected for and normalizedto background fluorescence ΔF/F=(F−F₀)/F₀ and baseline noise wassubtracted. Results for the respective hTAS2Rs are shown in Table 1through 4 below.

TABLE 1 Response of hTAS2R1 on chloramphenicol, humulone,diphenylthiourea, picrotoxinin and yohimbine max. concen- thres- ampli-com- tration hold EC₅₀ tude pound structure mock signal (mM) (mM) (mM)(counts) Chloram- phenicol

1 0.3 0.1 0.03 ~0.1 0.2 9000 Humu- lone

0.0003 0.0001 0.00003 ~0.0001 0.0009 9000 Di- phenyl- thiou- rea

1.0 0.3 0.1 0.03 ~0.1 — 4000 Picro- toxinin

0.001 0.0001 0.000001 ~0.0001 no data 2000 Yohim- bine

0.1 0.03 0.01 ~0.03 no data 3000 Scale denotes 1 min and 2000 counts.

TABLE 2 Response of hTAS2R3 on creatinine max. concentration thresholdEC₅₀ amplitude compound structure mock signal (mM) (mM) (mM) (counts)Creatinine

150 100  30 ~30 not determ. 5000 Scale denotes 1 min and 1000 counts.

TABLE 3 Response of hTAS2R7 on creatinine and cromolyn concen- max.tration threshold EC₅₀ amplitude compound structure mock signal (mM)(mM) (mM) (counts) Creatinine

150 100  30 ~10 not determ. 3000 Cromolyn

30 10  3 ~1  9 3000 Scale denotes 1 min and 1000 counts.

TABLE 4 Response of hTAS2R40 on humulone concen- max. tration thresholdEC₅₀ amplitude compound structure mock signal (mM) (mM) (mM) (counts)Humulone

0.0001 0.00001 0.00003 <0.00003 not determ. 6000 Scale denotes 1 min and2000 counts.

1. A method for isolating an agonist or antagonist of bitter tastereceptor activity comprising: (a) contacting: (i) a polypeptide with apotential antagonist; or (ii) a cell expressing a polypeptide with apotential antagonist; wherein said polypeptide according to (i) or (ii)has bitter taste receptor activity and wherein said polypeptideaccording to (i) or (ii): (1) comprises SEQ ID NO: 6; or (2) comprises apolypeptide containing only conservative amino acid substitutions in theamino acid sequence of SEQ ID NO: 6, said conservative amino acidsubstitutions numbering 15 or fewer conservative amino acidsubstitutions in the amino acid sequence of SEQ ID NO: 6; and (b)determining whether the potential antagonist antagonizes the bittertaste receptor activity of said polypeptide; wherein prior,concomitantly and/or after step (a) said polypeptide according to (i) orsaid cell according to (ii) is contacted with an agonist selected fromthe group consisting of chloramphenicol, humulone, diphenylthiourea,picrotoxinin and agonistic derivatives thereof.
 2. The method of claim1, further comprising admixing the identified antagonist with foodstuffsor any precursor material or additive employed in the production offoodstuffs.
 3. The method of claim 1, further comprising formulating theantagonist with an active agent in a pharmaceutically acceptable form.4. The method according to claim 1, wherein said polypeptide comprisesSEQ ID NO: 6 and said polypeptide has bitter taste receptor activity. 5.The method according to claim 1, wherein said method comprisescontacting a cell expressing a polypeptide comprising SEQ ID NO: 6, saidpolypeptide having bitter taste receptor activity.
 6. The methodaccording to claim 1, wherein said method comprises contacting a cellexpressing a polypeptide containing only conservative amino acidsubstitutions in the amino acid sequence of SEQ ID NO: 6, saidconservative amino acid substitutions numbering 15 or fewer conservativeamino acid substitutions in the amino acid sequence of SEQ ID NO:
 6. 7.The method according to claim 1, wherein said method comprisescontacting a cell expressing a polypeptide containing only conservativeamino acid substitutions in the amino acid sequence of SEQ ID NO: 6,said conservative amino acid substitutions numbering 14 or fewerconservative amino acid substitutions in the amino acid sequence of SEQID NO:
 6. 8. The method according to claim 1, wherein said methodcomprises contacting a cell expressing a polypeptide containing onlyconservative amino acid substitutions in the amino acid sequence of SEQID NO: 6, said conservative amino acid substitutions numbering 10 orfewer conservative amino acid substitutions in the amino acid sequenceof SEQ ID NO:
 6. 9. The method according to claim 1, wherein said methodcomprises contacting a cell expressing a polypeptide containing onlyconservative amino acid substitutions in the amino acid sequence of SEQID NO: 6, said conservative amino acid substitutions numbering 8 orfewer conservative amino acid substitutions in the amino acid sequenceof SEQ ID NO:
 6. 10. The method according to claim 1, wherein saidmethod comprises contacting a cell expressing a polypeptide containingonly conservative amino acid substitutions in the amino acid sequence ofSEQ ID NO: 6, said conservative amino acid substitutions numbering 5 orfewer conservative amino acid substitutions in the amino acid sequenceof SEQ ID NO:
 6. 11. The method according to claim 1, wherein saidmethod comprises contacting a cell expressing a polypeptide containingonly conservative amino acid substitutions in the amino acid sequence ofSEQ ID NO: 6, said conservative amino acid substitutions numbering 3 orfewer conservative amino acid substitutions in the amino acid sequenceof SEQ ID NO:
 6. 12. The method according to claim 1, wherein saidmethod comprises contacting a polypeptide containing only conservativeamino acid substitutions in the amino acid sequence of SEQ ID NO: 6,said conservative amino acid substitutions numbering 15 or fewerconservative amino acid substitutions in the amino acid sequence of SEQID NO:
 6. 13. The method according to claim 1, wherein said methodcomprises contacting a polypeptide containing only conservative aminoacid substitutions in the amino acid sequence of SEQ ID NO: 6, saidconservative amino acid substitutions numbering 14 or fewer conservativeamino acid substitutions in the amino acid sequence of SEQ ID NO:
 6. 14.The method according to claim 1, wherein said method comprisescontacting a polypeptide containing only conservative amino acidsubstitutions in the amino acid sequence of SEQ ID NO: 6, saidconservative amino acid substitutions numbering 10 or fewer conservativeamino acid substitutions in the amino acid sequence of SEQ ID NO:
 6. 15.The method according to claim 1, wherein said method comprisescontacting a polypeptide containing only conservative amino acidsubstitutions in the amino acid sequence of SEQ ID NO: 6, saidconservative amino acid substitutions numbering 8 or fewer conservativeamino acid substitutions in the amino acid sequence of SEQ ID NO:
 6. 16.The method according to claim 1, wherein said method comprisescontacting a polypeptide containing only conservative amino acidsubstitutions in the amino acid sequence of SEQ ID NO: 6, saidconservative amino acid substitutions numbering 5 or fewer conservativeamino acid substitutions in the amino acid sequence of SEQ ID NO:
 6. 17.The method according to claim 1, wherein said method comprisescontacting a polypeptide containing only conservative amino acidsubstitutions in the amino acid sequence of SEQ ID NO: 6, saidconservative amino acid substitutions numbering 3 or fewer conservativeamino acid substitutions in the amino acid sequence of SEQ ID NO: 6.